Since its invention centuries ago, light microscopy has evolved through many incarnations with distinct contrast mechanisms and hardware implementations. However, the fundamental motivation for its use has remained the same—it can resolve features that are not distinguishable by the naked eye. As a result, the push for higher resolution has been the focus of light microscopy development in recent years and several methods have been demonstrated to break the diffraction limit of conventional light microscopy. Despite all these efforts, one often underappreciated fact remains: for many biological samples, diffraction-limited resolution is rarely achieved, even for high-end research microscopes. Ideal imaging performance of a light microscope requires the excitation and/or emission light to pass through samples with optical properties identical to those of the designed immersion media, and any deviation from such conditions causes optical distortions, known as aberrations, leading to the loss of signal, image fidelity, and resolution. In practice, biological samples have inhomogeneous optical properties, so that images are increasingly degraded with increasing depth within biological tissues. For example, in point-scanning microscopes such as a two-photon fluorescence microscope, the aberrations of the excitation light result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution.
Accordingly, there exists a need for systems and methods to address the shortfalls of present technology and to provide other new and innovative features.